Occupancy of platelet receptors for platelet-activating factor in asthmatic patients during an allergen-induced bronchoconstrictive reaction.

The administration of platelet-activating factor (PAF) to human subjects triggers asthma-like responses. We investigated whether a bronchoconstrictive reaction was accompanied by the release of PAF in the circulation of allergic asthmatics. The appearance of PAF was assessed by measuring the number of freely accessible PAF-receptors on platelets in vitro, assuming that the contact between platelets and PAF in vivo would reduce the receptor binding of [3H]PAF in vitro. 16 asthmatics were challenged twice, first with buffer and the next day with allergen. A comparison between receptor binding after provocation with the data of the same patients after allergen challenge revealed a significant difference in PAF binding (P = 0.034), with an average decrease of 14% immediately after allergen challenge followed by a return to control values after about 4 h and a transient increase of 9% at 7 h after provocation. The decrease in accessible PAF receptors was accompanied by a slight decrease in platelet count in peripheral blood between 30 min and 4 h after allergen challenge. The platelet counts recovered to the original values afterwards. These data support the concept that in patients with allergic asthma PAF is secreted in the circulation. The contact between PAF and the platelets may trigger the transient sequestration of platelets, possibly in the lung. Thus, PAF and platelets may contribute to the pathogenesis of allergic asthma.

Late asthmatic responses after exercise challenge are reproducible.

In this study the reproducibility of a late asthmatic reaction (LAR) after exercise challenge (EC) has been documented. Eighty-three hospitalized patients with asthma were challenged with exercise. The patients were examined according to a standardized protocol that comprised 8 minutes of bicycling at 90% of predicted heart rate. An LAR after EC was considered to have occurred when there was a fall in peak expiratory flow rate greater than or equal to 20% on three or more time points on the exercise day compared to corresponding clock time on a control day. According to these criteria, 11 patients (13.3%) experienced an LAR. Those patients were rechallenged 21 to 150 days after the first EC, without changing the therapy regimen of the patients, to study its reproducibility. Eight patients (73%) demonstrated a reproducible LAR after EC based on the criteria for a positive LAR. Although the LAR after EC was reproducible, the time points at which the LAR took place after the second EC differed from LARs after the first EC. Our results indicate that the LAR after EC occurs in a considerable number of patients with bronchial asthma and is quite reproducible.

Leukotriene C4 formation by purified human eosinophils can be induced by arachidonic acid in the absence of calcium-ionophore A23187.

Addition of arachidonic acid (50 microM) to purified human eosinophils leads to the formation of considerable amounts of LTC4 [11.3 +/- 1.3) x 10(6) molecules/cell, mean +/- SEM, n = 10), 15-HETE [412 +/- 142) x 10(6) molecules/cell, mean +/- SEM, n = 3) and 15-series leukotrienes [35 +/- 15) x 10(6) molecules/cell, mean +/- SEM, n = 3). The ratio of the amounts of LTC4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. Platelet activating factor (1 microM) was able to enhance significantly the production of LTC4 but not that of 15-lipoxygenase products. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains, at least partly, its ability to induce the Ca2+-dependent formation of LTC4.

The possible role of particular leukotrienes in the allergen-induced late-phase asthmatic reaction.

Challenge with allergen may provoke an early-phase and late-phase asthmatic reaction in allergic asthmatic individuals. Although the pathogenesis of the allergen-induced late-phase asthmatic reaction (LAR) is still poorly understood, the cell types and mediators involved in this reaction are considered to be of extreme importance for the understanding of the pathogenesis of asthma. Evidence has been provided that predominantly eosinophils penetrate into the bronchioli at the beginning of the LAR. The mobilization of those eosinophils into the lung tissue is caused by the release of chemotactic factors of protein or lipid nature, most likely platelet activating factor (PAF). How these cells are finally activated to release their mediators, when they have infiltrated into the bronchioli, remains obscure. They may, however, actively contribute to the occurrence of the LAR by the release of the strongly bronchoconstrictive compound leukotriene C4 (LTC4). Our investigations have shown that eosinophils do possess the capacity to synthesize this mediator upon in-vitro challenge with the calcium ionophore A23187, zymosan particles coated with IgG and C3b (C3bi) or PAF at relatively high concentrations. As to demonstrating the possible in-vivo formation of leukotrienes, studies have been undertaken to demonstrate the excretion of a stable metabolite of LTC4, i.e. LTE4, into human urine. In our first series of investigations normal individuals were challenged via inhalation of high amounts of LTD4. Besides a bronchoconstrictive reaction and an increase in the level of maximal airway narrowing to challenge with methacholine, significant LTE4 excretion could be demonstrated in the urine. Similar studies concerning allergen provocation in allergic asthmatic individuals are now in progress.(ABSTRACT TRUNCATED AT 250 WORDS)

Associated expression of CD1 antigen and Fc receptor for IgE on epidermal Langerhans cells from patients with atopic dermatitis.

The presence of Fc receptors for IgE on epidermal Langerhans cells (LC) from patients with atopic dermatitis (AD) was demonstrated by three different types of experiments. Firstly, cell-bound IgE on LC was removed by acid elution and restored by highly purified human myeloma IgE (IgE kappa). Secondly, after pepsin digestion of cell-bound IgE the number of LC staining with anti-human light chain (kappa, lambda) antibodies significantly decreased in contrast to the number of LC staining with anti-human epsilon heavy chain antibody. Thirdly, LC formed rosettes with sheep red blood cells (SRBC) coated with IgE kappa. Epidermal LC from normal non-atopic controls, did not form rosettes with SRBC-IgE. The SRBC-IgE rosette formation could be inhibited by preincubation with IgE kappa and BB10 (MoAb directed against the Fc receptor for IgE on human eosinophils, platelets and macrophages), but also with human IgG, whereas the SRBC-IgG rosette formation could be inhibited neither by IgE kappa nor by BB10. Both the SRBC-IgE and the SRBC-IgG rosette formation could be inhibited by OKT6 (anti-CD1) antibody. The results of inhibition studies with OKT6 antibody on the reconstitution of IgE on epidermal LC after acid elution suggest an associated expression of the CD1 antigen and the Fc receptor for IgE.

Arachidonic acid can induce leukotriene C4 formation by purified human eosinophils in the absence of other stimuli.

Stimulation of purified human eosinophils with 50 microM arachidonic acid leads to the production of leukotriene C4, 15-hydroxy-eicosatetraenoic acid and 15-series leukotrienes. The ratio of the amounts of leukotriene C4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. In the presence of 1 microM platelet-activating factor a significant elevation of leukotriene C4 formation is observed, whereas the formation of 15-lipoxygenase products remains unaltered. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains at least partly its ability to induce the Ca2+-dependent formation of leukotriene C4.

Active participation of eosinophils in patch test reactions to inhalant allergens in patients with atopic dermatitis.

Intracutaneous testing and patch tests with house dust mite and grass pollen allergens were performed in patients with atopic dermatitis. Only patients with an immediate type skin reaction to house dust mite or grass pollen allergens showed a positive patch test reaction to these allergens 24-48 h after testing. Occasionally positive patch test reactions at 20 min, 2 h and 6 h were also observed. Patch test reactions were not found in normal controls or atopic patients without atopic dermatitis. Analysis of the cellular infiltrate demonstrated an influx of eosinophils into the dermis, starting from 2-6 h after patch testing. Immunostaining with antibodies against granular constituents of the eosinophils revealed that the infiltrating eosinophils were in an activated state and had lost part of their granular contents. At 24 h eosinophils also appeared in the epidermis. Electron microscopy showed that in the epidermis, some eosinophils were in close contact with Langerhans cells, suggesting a cell-cell interaction. Taken together, these results strongly suggest an active role for eosinophils in patch test reactions to inhalant allergens in atopic dermatitis patients.

The excretion of leukotriene E4 into urine following inhalation of leukotriene D4 by human individuals.

Healthy volunteers underwent bronchial challenge with increasing doses of nebulized leukotriene D4 (0.007 - 200 nmol) at 15 min intervals. Total amounts of 200 nmol (females) and 400 nmol (males) were inhaled, corresponding to approximately 100 nmol and 200 nmol deposited in the lung, respectively. Of the latter amounts 3 +/- 1% (mean +/- S.E.M., n = 5) was found to be excreted as leukotriene E4 into the urine within 12 h. No further excretion after this period was observed. Approximately 50% of the total urinary leukotriene E4 was excreted during the first 2 h. These results suggest that a possible formation of sulfidopeptide leukotrienes in the lung in vivo can be monitored by measuring leukotriene E4 excretion into the urine.

Birch pollen asthma in The Netherlands.

The occurrence of birch pollen asthma is related to high birch pollen concentrations in the air, and therefore this type of asthma is rather common in Scandinavia. High birch pollen concentrations are rare in The Netherlands, but in the spring of 1984 extremely high levels were reached, whereas the concentrations of alder and hazel pollen were very low. During this period nine pollinosis patients known to the allergist developed asthmatic complaints. The appearance and disappearance of the asthmatic symptoms in these patients showed strong correlation with the course of the birch pollen concentration in the air, which strongly suggests that birch pollen asthma can occur in The Netherlands. The significance of this finding may be that this type of asthma can be observed regularly when the birch pollen concentrations are very high. This may also hold for other western European countries.

Platelet-activating factor (PAF-acether) induced leukotriene C4 formation and luminol dependent chemiluminescence by human eosinophils.

Human eosinophils are capable of synthesizing almost exclusively the strongly spasmogenic compound LTC4 when stimulated with either the calcium ionophore A 23187 or opsonized zymosan (OZ). Although PAF-acether in concentrations ranging from 10 nM to 1 microM is hardly capable of inducing significant LTC4 synthesis itself, it significantly enhances the OZ-induced LTC4 formation at a concentration of 1 microM. However, at a concentration of 10 microM, PAF-acether itself is capable of inducing LTC4 formation comparable with that induced by OZ. PAF-acether, at a concentration of 10 microM (and not at a concentration of 1 microM) is also capable of inducing a luminol dependent chemiluminescent response by eosinophils. The PAF-acether antagonist BN 52021 at a concentration of 0.1 mM not only partially inhibited the PAF-acether induced LTC4 formation but also the OZ induced LTC4 formation. Since an equal inhibition is found the inhibitory mode of action of BN 52021 is most likely directed towards a common pathway. Taken together, these results suggest that eosinophils may be triggered by high locally reached concentrations of PAF-acether to release inflammatory and bronchoconstrictive mediators. This may be of importance for the pathogenesis of the allergen induced late phase asthmatic reaction.

The presence of IgE molecules on epidermal Langerhans cells in patients with atopic dermatitis.

Skin sections of clinically involved and clinically normal-looking skin from patients with atopic dermatitis were incubated with anti-human IgE antibodies using the indirect immunoperoxidase technique. Apart from positive dermal anti-IgE staining, positive epidermal anti-IgE staining was also observed. The morphology of the epidermal staining cells suggested the involvement of dendritic cells. This was confirmed by positive immuno-double labelling with OKT6 and anti-IgE. This phenomenon seemed to be specific for atopic dermatitis since skin sections from normal non-atopic controls, patients with allergic asthma, contact dermatitis, and schistosomiasis showed no epidermal anti-IgE staining. To further elucidate the nature of the epidermal anti-IgE staining cells, epidermal cell suspensions were prepared from clinically involved skin from patients with atopic dermatitis. These cell suspensions also showed positive anti-IgE staining cells and positive immuno-double labelling with OKT6 and anti-IgE. Immunogold electron microscopy with anti-IgE on epidermal cell suspensions from patients with atopic dermatitis showed gold particles on the cell membranes of cells containing Birbeck granules, being Langerhans' cells. Epidermal cell suspensions from normal non-atopic controls were negative. The presence of IgE molecules on epidermal Langerhans' cells, which seems to be specific for patients with atopic dermatitis, provides an explanation for the high frequency of positive patch test reactions to inhalant allergens.

Leukotrienes and their possible significance for the pathogenesis of asthma.

Six years ago the structure of slow reacting substance of anaphylaxis (SRS-A), a strongly bronchoconstrictive substance, has been unravelled SRS-A proved to be a mixture of different closely related compounds, now denominated as sulfidopeptide Leukotrienes. Leukotrienes possess a conjugated triene system and one or more oxygen functions. They are formed from membrane derived arachidonic acid by an initial oxygenation by the enzyme lipoxygenase. Sofar the following leukotrienes have been characterized leukotriene A4, B4, C4, D4, E4 and F4. Leukotrienes possess important biological properties. Leukotriene B4 is strongly chemotactic for leukocytes, whereas the sulfidopeptide leukotrienes C4, D4 and E4 are strongly spasmogenic. In this review the formation and the different biological activities of leukotrienes and the possible role of leukotrienes in the asthmatic process will be discussed.

On the optimal conditions of LTC4 formation by human eosinophils in vitro.

The optimal conditions for the -in vitro- LTC4 formation by the human eosinophil, isolated from peripheral blood, have been investigated in detail. LTC4 formation was found strongly Ca2+ and ionophore dependent and was complete after 20 min. Maximal LTC4 production was observed in the presence of 2 mM Ca2+, 10 microM ionophore A23187 and 5 mM glutathione. Addition of arachidonic acid resulted in a significant inhibition of the LTC4-synthesis by human eosinophils. In contrast, the formation of 15-HETE was strongly stimulated by the addition of arachidonic acid. As the LTC4 synthesis was found to be strongly inhibited by the addition of 15(S)-HETE to the incubation medium, this monohydroxy acid may be responsible for the inhibitory activity of arachidonic acid.

Some fundamental points concerning the clinical aspects of desensitization.

Continued exposure of target cells to a hormonal agonist frequently leads to a blunted response to that agonist. This process has been termed desensitization, refractoriness, tolerance or tachyphylaxis. The receptor involved in case of beta-sympathomimetics is the beta-adrenergic receptor. When the clinical aspects of beta-adrenergic receptor desensitization are considered, a number of points need serious attention: 1) the route of administration of the drug and the concentration of the drug reached at the receptor site; 2) other factors which may cause reduced responsiveness of the beta-adrenergic receptor besides beta-sympathomimetics and interfere with this process; 3) the different cell-types which are studied and thought to be representative for processes taking place at lung tissue level; 4) the in vivo parameter(s) chosen to describe desensitization; 5) the difference between normal and asthmatic subjects; 6) the mechanism behind desensitization. The paper concerns a review of these points. Since the clinical findings with respect to the induction of desensitization by beta-sympathomimetics are rather controversial, the therapeutic advice should be: use it with caution.

Exclusive leukotriene C4 synthesis by purified human eosinophils induced by opsonized zymosan.

Purified human eosinophils were challenged with N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating-factor, valyl-glycyl-seryl-glutamic acid, phorbol myristate acetate, zymosan, opsonized zymosan and the calcium ionophore A23187 to induce leukotriene synthesis. Reversed-phase high performance liquid chromatography analysis demonstrated the almost exclusive synthesis of leukotriene C4 by eosinophils of 11 healthy donors after challenge with opsonized zymosan [(22 +/- 4) X 10(6) molecules LTC4/cell, mean +/- SE] or the calcium ionophore A23187 [(54 +/- 7) X 10(6) molecules LTC4/cell, mean +/- SE]. The other agents were not capable of inducing leukotriene formation. When in addition to opsonized zymosan N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor were added a significant increase of the leukotriene C4 synthesis by eosinophils was observed. These results suggest that eosinophils might be triggered to produce considerable amounts of the spasmogenic leukotriene C4 in vivo by C3b- and/or IgG-mediated mechanisms e.g. phagocytosis.

Measurement of total IgE antibody levels in lacrimal fluid of patients suffering from atopic and non-atopic eye disorders. Evidence for local IgE production in atopic eye disorders?

Total IgE levels in lacrimal fluid of patients suffering from different eye disorders were quantitatively measured by a modification of the paper radio immuno sorbent test (PRIST). The geometric mean values for patients with atopic conjunctivitis, patients with keratoconjunctivitis vernalis, and patients with asthma without conjunctivitis differed significantly from those for control persons and those for patients without atopic conjunctivitis. Besides lacrimal fluid IgE levels, serum IgE levels as well as lacrimal fluid and serum albumin levels were measured. From these values the local IgE production was calculated. Although there seemed a good correlation between the level of lacrimal fluid IgE and the amount of local IgE production, the results suggest that local IgE production in lacrimal fluid is not restricted to patients with atopic eye disorders only.